System and method for longitudinal analysis of peptide synthesis

ABSTRACT

The present invention provides a system and method for assessing a synthetic peptide population including interrogating a population of peptide features in the presence of a receptor having an affinity for a binder sequence. The population of peptide features is synthesized over a plurality of synthesis periods and includes a plurality of control peptide features synthesized to have an amino acid sequence including the binder sequence. The control peptide features include a first feature synthesized beginning with a first one of the synthesis periods, and a second feature synthesized beginning after the first one of the synthesis periods such that synthesis of the second control peptide feature is delayed by at least one synthesis period. The method further includes detecting a signal output characteristic of an interaction of the receptor with the control peptide features, the signal output indicative of the fidelity of synthesis of the population of peptide features.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is based on, claims the benefit of, and incorporatesherein by reference U.S. Provisional Patent Application Ser. No.62/247,485 filed on 28 Oct. 2015 and entitled, “System and Method forLongitudinal Analysis of Peptide Synthesis.”

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

Not applicable.

BACKGROUND OF THE INVENTION

The disclosure relates, in general, to evaluating peptide synthesis and,more particularly, to a system and method for identifying andimplementing quality control oligopeptide sequences for assayinglongitudinal peptide synthesis fidelity.

Peptides are biological polymers assembled, in part, through theformation of amide bonds between amino acid monomer units. In general,peptides may be distinguished from their protein counterparts based onfactors such as size (e.g., number of monomer units or molecularweight), complexity (e.g., number of peptides, presence of coenzymes,cofactors, or other ligands), and the like. Experimental approaches forthe identification of binding motifs, epitopes, mimotopes, diseasemarkers, or the like may successfully employ peptides instead of largeror more complex proteins that may be more difficult to obtain ormanipulate. As a result, the study of peptides and the capability tosynthesize those peptides are of significant interest in the biologicalsciences and medicine.

Several methods exist for the synthesis of peptides including both invivo and in vitro translation systems, as well as organic synthesisroutes such as solid phase peptide synthesis. Solid phase peptidesynthesis is a technique in which an initial amino acid is linked to asolid surface such as a bead, a microscope slide, or another likesurface. Thereafter, subsequent amino acids are added in a step-wisemanner to the initial amino acid to form a peptide chain. Because thepeptide chain is attached to a solid surface, operations such as washsteps, side chain modifications, cyclization, or other treatment stepsmay be performed with the peptide chain maintained in a discretelocation.

Recent advances in solid phase peptide synthesis have led to automatedsynthesis platforms for the parallel assembly of millions of uniquepeptide features in an array on a single surface (e.g., a ˜75 mm×˜25 mmmicroscope slide). The utility of such peptide arrays is, at least inpart, dependent on the accuracy and fidelity with which the synthesis iscarried out. For example, if the reagents used for synthesis aredegraded, contaminated or improperly transported to the array surfaceduring synthesis, a given peptide feature may have an altered,incomplete, or truncated peptide sequence. Other errors in peptidesynthesis may also occur. However, it is generally impractical withcurrently available technologies to assay the quality of everyindividual feature on a routine basis due to both the number of featuressynthesized on a given array, and the associated material masssynthesized for each feature.

Accordingly, there is a need for improved processes and systems for theanalysis of synthesis fidelity for peptide arrays as well as for peptidesynthesis in general.

SUMMARY OF THE INVENTION

The present invention overcomes the aforementioned drawbacks byproviding a system and method for analysis of peptide synthesisfidelity.

In accordance with one embodiment of the present disclosure, a method ofassessing a synthetic peptide population includes interrogating apopulation of peptide features in the presence of a receptor having anaffinity for a binder sequence. The population of peptide features issynthesized over a plurality of synthesis periods, and includes aplurality of control peptide features synthesized to have an amino acidsequence including the binder sequence. The plurality of control peptidefeatures includes a first control peptide feature synthesized beginningwith a first one of the plurality of synthesis periods, and a secondcontrol peptide feature synthesized beginning after the first one of theplurality of synthesis periods such that synthesis of the second controlpeptide feature is delayed by at least one synthesis period. The methodfurther includes detecting a signal output characteristic of aninteraction of the receptor with the plurality of control peptidefeatures, the signal output indicative of the fidelity of synthesis ofthe population of peptide features.

In one aspect, each of the plurality of synthesis periods comprises aplurality of synthesis cycles, wherein each of the plurality ofsynthesis cycles corresponds to the addition of a selected amino acid.

In another aspect, the binder sequence is a streptavidin bindersequence, and the receptor is streptavidin.

In yet another aspect, the plurality of control peptides issynthesizable over a minimum number of synthesis periods, and at least aportion of the plurality of control peptides is synthesized over anumber of synthesis periods greater than the minimum number of synthesisperiods. Further, in some embodiments the minimum number of synthesisperiods is at least two synthesis periods.

In a further aspect, the method includes contacting the population ofpeptide features in the presence of the receptor with a fluorescentprobe capable of binding to the receptor. The signal output is afluorescence intensity obtained through fluorophore excitation-emission,the fluorescence intensity reflecting at least one of an abundance of aportion of the receptor associated with the plurality of control peptidefeatures and a binding affinity of the receptor to the plurality ofcontrol peptide features.

In one aspect, the population of peptide features is covalently bound toa solid surface in an array. In some embodiments, the peptide featuresare bound to the solid surface at a density of at least about 100,000features per square centimeter.

In yet another aspect, the output signal of the receptor is known foreach of the plurality of binder sequences.

In still another aspect, the population of peptide features is preparedusing maskless array synthesis.

In still another aspect, the control peptide features are synthesized tohave at least a first amino acid sequence including a first bindersequence and a second amino acid sequence including a second bindersequence different from the first binder sequence. The receptor has anaffinity for each of the first binder sequence and the second bindersequence.

In accordance with another embodiment of the present disclosure, amethod of assessing the fidelity of a synthetic peptide populationincludes synthesizing a population of peptide features on a solidsurface over a plurality of sequential synthesis periods. The populationof peptide features includes a plurality of sample peptide features anda plurality of control peptide features synthesized to have an aminoacid sequence including a binder sequence. The control peptide featuresinclude a first control peptide feature synthesized beginning with afirst one of the plurality of synthesis periods, and a second controlpeptide feature synthesized beginning after the first one of theplurality of synthesis periods such that synthesis of the second controlpeptide feature is delayed by at least one synthesis period. The methodfurther includes contacting the population of peptide features on thesolid surface with a receptor having an affinity for the bindersequence, and detecting an output characteristic of an interaction ofthe receptor with each of the control peptide features. The output isindicative of the longitudinal fidelity of synthesis of the populationof peptide features.

In one aspect, each of the plurality of synthesis periods comprises aplurality of synthesis cycles, and each of the plurality of synthesiscycles corresponds to the addition of a selected amino acid.

In another aspect, the binder sequence is a streptavidin bindersequence, and the receptor is streptavidin.

In yet another aspect, the plurality of control peptides issynthesizable over a minimum number of synthesis periods, and wherein atleast a portion of the plurality of control peptides is synthesized overa number of synthesis periods greater than the minimum number ofsynthesis periods.

In a further aspect, the method includes contacting the population ofpeptide features in the presence of the receptor with a fluorescentprobe capable of binding to the receptor. The signal output is afluorescence intensity obtained through fluorophore excitation-emission,the fluorescence intensity reflecting at least one of an abundance of aportion of the receptor associated with the plurality of control peptidefeatures and a binding affinity of the receptor to the plurality ofcontrol peptide features.

In still another aspect, each of the sample peptide features has adefined sequence. In some embodiments, the peptide features are bound tothe solid surface at a density of at least about 100,000 features persquare centimeter.

In one aspect, the output signal of the receptor is known for each ofthe plurality of binder sequences.

In another aspect, the population of peptide features is prepared usingmaskless array synthesis.

In still another aspect, the control peptide features are synthesized tohave at least a first amino acid sequence including a first bindersequence and a second amino acid sequence including a second bindersequence different from the first binder sequence. The receptor has anaffinity for each of the first binder sequence and the second bindersequence.

In accordance with a further embodiment of the present disclosure, asynthetic peptide array includes an array substrate comprising a solidsupport having a reactive surface, and a population of peptide featuresimmobilized on the reactive surface. The population of peptide featuresis synthesized over a plurality of sequential synthesis periods andincludes a plurality of control peptide features synthesized to have anamino acid sequence including a binder sequence. The plurality ofcontrol peptide features includes a first control peptide featuresynthesized beginning with a first one of the plurality of synthesisperiods, and a second control peptide feature synthesized beginningafter the first one of the plurality of synthesis periods such thatsynthesis of the second control peptide feature is delayed by at leastone synthesis period. Detecting a signal output characteristic of aninteraction of a receptor with each of the control peptide features isindicative of the fidelity of synthesis of the population of peptidefeatures.

In one aspect, the binder sequence is a streptavidin binder sequence,and the receptor is streptavidin.

The foregoing and other aspects and advantages of the invention willappear from the following description. In the description, reference ismade to the accompanying drawings which form a part hereof, and in whichthere is shown by way of illustration a preferred embodiment of theinvention. Such embodiment does not necessarily represent the full scopeof the invention, however, and reference is made therefore to the claimsand herein for interpreting the scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration showing a partial plan view of apeptide array including a plurality of control peptide featuresaccording to the present disclosure.

FIG. 2 is a partial elevational view of a peptide array similar to thepeptide array of FIG. 1 at an intermediate time point during synthesisof the control peptide features.

FIG. 3 is a table showing a design scheme for a first example peptidearray synthesis scheme that includes nine sequential synthesis periods,where each period includes twenty cycles, with one cycle allocated foreach one of the twenty canonical amino acids.

FIG. 4 is a table similar to FIG. 3 showing a design scheme for a secondexample peptide array synthesis scheme.

FIG. 5 is a schematic illustration of an embodiment of a peptide arrayincluding a population of peptide features for the interrogation anddetection of control peptide features.

FIG. 6 is a schematic illustration of the peptide array of FIG. 5following exposure of the control peptide features to a plurality ofreceptor molecules.

FIG. 7 is a schematic illustration of the peptide array of FIG. 6following binding of a detectable tag to the receptor molecules.

FIG. 8 is a schematic illustration showing a partial plan view of anembodiment of a peptide array including a plurality of uniformlysynthesized control peptide features.

FIG. 9 is an example plot of signal output for each of the controlpeptide features in the peptide array of FIG. 7.

FIG. 10 is a schematic illustration showing a partial plan view of anembodiment of a peptide array including a plurality of synthesizedcontrol peptide features exhibiting longitudinal variability.

FIG. 11 is an example plot of signal output for each of the controlpeptide features in the peptide array of FIG. 9.

FIG. 12 is an example plot showing fluorescence signal output for aplurality of control peptide features from four separate synthesisoperations as a function of synthesis period.

FIG. 13 is an example of a method for assessing the longitudinalfidelity of a synthetic peptide population according to the presentdisclosure.

Like numbers will be used to describe like parts from Figure to Figurethroughout the following detailed description.

DETAILED DESCRIPTION OF THE INVENTION I. Overview

As also discussed above, in various situations it may be useful toprovide quality control measures for assessing the fidelity of aplurality of synthetic peptides. Herein the terms fidelity and qualityare used to mean the accuracy with which the desired sequence isreplicated by synthesis. Accordingly, a synthesized peptide or peptidefeature having high fidelity or quality has a peptide sequence that issubstantially identical to a predefined or desired sequence with noinsertions, substitutions, deletions, additions, or other likemodifications. By contrast, a synthesized peptide or peptide featurehaving low fidelity or quality has a peptide sequence that includes oneor more insertions, substitutions, deletions, additions, or other likemodifications relative to the predefined or desired sequence. Withrespect to assessing the fidelity of a plurality of synthetic peptides,in one example, it may be useful to check for successful incorporationof each type of amino acid or other monomer unit used in the synthesisof one or more peptide features in a solid phase peptide synthesisoperation. In another example, it may be useful to monitor the qualityof reagents used for solid phase peptide synthesis along with anyassociated process equipment for delivery of the reagents. In yetanother example, it may be useful to determine the overall quality of anarray in a non-destructive manner, by analyzing only a small subset ofpeptides, the like, or combinations thereof. In still another example,it may be useful to monitor the quality of synthesis over time to ensureuniform synthesis quality from the beginning to the end of a particularpeptide synthesis operation. Accordingly, many peptide synthesis schemesinclude various quality control sequences or analysis schemes to checkfor synthesis.

In one aspect, current quality control measures may pose severalproblems. For example, U.S. Pat. No. 6,955,915 to Fodor et al. describesa quality control method in which an initial binding profile may bemeasured for a fixed array design. Thereafter, binding profiles may beobtained for subsequent arrays of the same design for comparison withthe initial binding profile. One challenge associated with this approachis that a new binding profile may need to be prepared for each uniquearray design. Further, a change in binding profiles between samples maynot be informative as to the cause of the change. Yet other qualitycontrol methods may only indicate the general occurrence of an error, orin some limited cases (e.g., vertical tiling in oligonucleotide arrays),the occurrence of an error during a particular synthesis cycle.Moreover, it may be difficult to track changes in the quality orfidelity of synthesis across both synthesis cycles and synthesis periods(i.e., the longitudinal quality or fidelity). Ultimately, theaforementioned quality control methods do not enable tracking of eitherthe particular cause of a synthesis error, or the longitudinal qualityor fidelity of peptide synthesis. Further challenges may arise dependingon the number of peptide features, the category of the solid surface(e.g., beads vs. arrays) upon which the synthesis is performed, the sizeor complexity of the synthesized peptide features, the duration of thepeptide synthesis operation (e.g., number of synthesis steps, overalltime, etc.), and the like.

These and other challenges may be overcome with a system and method forassessing longitudinal peptide synthesis fidelity according to thepresent disclosure. In one embodiment of the present disclosure, acontrol peptide feature having a particular binder sequence issynthesized multiple times over the course of synthesis of a broaderpopulation of peptide features. The quality of the control peptidefeatures over time, as assessed by binding to a receptor having anaffinity for the binder sequence, is used to determine the quality ofthe population of peptide features from a longitudinal or temporalperspective. Herein, the term “quality” refers to a measure of acharacteristic or aspect of the component or feature in question. Forexample, the quality of a peptide feature can include the fidelity withwhich the sequence of the peptides was synthesized or reproduced, thefraction of peptides within a peptide feature that possess the correctsequence, or the like. In one aspect, the quality of a peptide (orpeptide feature) may be determined by interrogating the interaction ofthe peptide with a receptor having a known affinity for a bindersequence included in the peptide. In another aspect, the term “drift”refers to the occurrence of longitudinal changes in synthesis quality orfidelity between synthesis cycles, synthesis periods, and combinationsthereof.

In one example, synthesis of the population of peptide features iscarried out over a number of synthesis periods, where each period isfurther divided into a plurality of synthesis cycles that correspond tothe addition of one or more amino acids or other synthesis reagents. Oneor more control peptide features are synthesized beginning with thefirst period, and the quality of the one or more control peptidefeatures are compared to subsequently synthesized control peptides wherethe initiation of synthesis was delayed by one or more synthesis cyclesor synthesis periods. Initially, a control peptide is selected that canbe completely synthesized over a minimum number of synthesis periods. Inone example, a control peptide can be completely synthesized over aminimum of three (consecutive or non-consecutive) synthesis periods. Fora synthesis run having a duration of twelve periods, initiation ofsynthesis of one or more of the selected control peptides may be delayedby up to (and including) nine periods, with the final set of controlpeptides synthesized over the final three periods (i.e., periods ten,eleven, and twelve). The synthesis run can then include control peptidefeatures synthesized beginning at one or more of the first tenconsecutive synthesis periods (of the twelve total synthesis periods)for longitudinal analysis of the quality of the broader population ofpeptide features.

In some embodiments, the quality of the synthesized control peptidefeatures is determined by assaying reporter-labeled receptor binding tothe control peptide features. In one example, receptor binding tocontrol peptide features synthesized beginning with the initialsynthesis period is compared to receptor binding to control peptidefeatures synthesized beginning after the initial synthesis period.Differences in the observed binding of the receptor for the differentcontrol peptide features (either higher or lower reporter output signal)is indicative of a change in the longitudinal quality of the controlpeptide features (i.e., drift), and by extension, the quality of theoverall population of peptide features. In one aspect, staggeringsynthesis of the control peptide features over the total number ofsynthesis periods enables longitudinal analysis of peptide synthesis.

With respect to timing of synthesis of the control peptides, the presentdisclosure provides for a variety of approaches. In some embodiments,each synthesis period includes a number of synthesis cycles or stepsthat are carried out in a fixed order. For example, synthesis of apopulation of peptide features using each of the twenty canonical aminoacids can be performed over a number of synthesis periods having twentycycles each, where each cycle corresponds to the addition of one of thetwenty canonical amino acids. The order of the cycles dictates theminimum number of periods required to synthesize a given control peptidesequence. For an example control peptide feature having a sequence thatcan be synthesized over a minimum of three synthesis periods, a numberof synthesis options exist. In a first approach, the control peptidefeature can be completely synthesized over three consecutive(sequential) synthesis periods. In a second approach, synthesis of thecontrol peptide feature can be distributed over three non-consecutivesynthesis periods. In a third approach, synthesis of the control peptidefeature can be distributed over greater than three (i.e., four or more)synthesis periods, which can be consecutive or non-consecutive. Notably,each of the aforementioned approaches can be used individually or incombination for synthesis of a plurality of control peptide featureswithin a broader population of peptide features. Further, synthesis ofcontrol peptide features can be initiated beginning in a first orsubsequent synthesis period such that the synthesis periods selected forsynthesis of one control peptide feature partially overlap, completelyoverlap, or do not overlap at all with the synthesis periods selectedfor synthesis of another control peptide feature.

The use of reporter-labeled receptors having an affinity for bindersequences within the control peptides leverages measuring a signaloutput characteristic of an interaction of a receptor with a particularpeptide sequence to detect an absolute or relative receptor affinity.The detected signal output can then be used to determine synthesisfidelity for a set of control peptides, and by extension, a broaderpopulation of peptides that includes the control peptides. In someembodiments, the aggregate data collected for a set of control peptidesis indicative of a particular synthesis error. For example,interrogating a plurality of control peptides synthesized at variousspatial locations across the array or at different times over the courseof a synthesis operation may result in a measurable change in binding ofthe control peptide by a receptor having an affinity for the uniquebinder sequence. The resulting measurements can be analyzed to determinethe likelihood of an occurrence of a substitution, deletion, or othersynthesis error that occurred during array synthesis. Moreover, thedesign approach for how the control peptide features are synthesized asdiscussed above can be selected to elucidate whether or not a particularerror or incident occurred during synthesis that affects the quality ofthe population of peptide features.

In summary, according to one embodiment of the present disclosure,successful synthesis of a population of peptides can be monitored by (i)characterizing a binder sequence-receptor pair where modification of thebinder sequence results in a measurable change for a characteristic ofan interaction (e.g., binding) of the receptor with the binder sequence,(ii) including in the population of peptides to be synthesized a controlpeptide having the characterized binder sequence, (iii) longitudinallystaggering synthesis of the control peptides over the course ofsynthesis of the broader population of peptide features, and (iv)detecting the characteristic of the interaction following synthesis ofthe population of peptides to determine the overall quality of thetemporally staggered control peptides, and by extension, the overallquality of the population of peptides, in general.

In one aspect, the present disclosure provides a method of assessing thelongitudinal quality of a synthetic peptide population. For the purposesof the present disclosure, a synthetic peptide population includes anyset of two or more peptides or peptide features (i.e., a grouping of twoor more peptides having the same monomer sequence) prepared in astep-by-step chemical synthesis operation. For example, a syntheticpeptide population may be prepared by solid phase peptide synthesis,where an initial amino acid is covalently bound to a solid surfaceeither directly or via one or more linker molecules. Thereafter,subsequent amino acids may be added to the initial amino acid indirected or random fashion in order to prepare a population of peptidefeatures arranged on a single surface such as a microscope slide, ordistributed across a plurality of beads or other particle supports. Oneparticular method for preparation of a population of synthetic peptidesincludes maskless array synthesis (MAS) technology (see, e.g., U.S. Pat.No. 8,658,572 to Albert et al.). However, other solid phase peptidesynthesis methods, which are well known in the art, may be used for theformation of a synthetic peptide population according to the presentdisclosure.

For assessment of the synthetic peptide population, a method may includea first step of interrogating a population of peptide features in thepresence of a receptor having an affinity for a plurality of bindersequences. A receptor includes any peptide, protein, antibody, smallmolecule, or other like structure that is capable of specificallybinding a given peptide sequence or feature. In general, an aspect ofthe receptor should be detectable in order to determine whether thereceptor is bound to a particular peptide or peptide feature. Forexample, the receptor itself may include a fluorophore that isdetectable with a fluorescence microscope. Alternatively (or inaddition), the receptor may be bound by a secondary molecule such as afluorescent antibody. Further approaches will also fall within the scopeof the present disclosure.

As described above the receptor is capable of binding to or otherwiseinteracting with a known binder sequence or affinity sequence. Oneexample of a binder sequence is a defined amino acid sequence or motif.The defined amino acid sequence can represent at least a portion of afull length peptide within the synthetic peptide population. However,the binder sequence can itself be a full length peptide. For example,the eight amino acid peptide sequence Trp-Ser-His-Pro-Gln-Phe-Glu-Lys(i.e., WSHPQFEK (SEQ ID NO:3)) known as a “Strep-tag” exhibits intrinsicaffinity towards an engineered form of the protein streptavidin.According to the present disclosure, a Strep-tag can be incorporated ateither the N-terminus or the C-terminus of a given peptide or evenincorporated at an intermediate point within a peptide. Thereafter, thepeptide population including the peptides consisting of (or comprising)the Strep-tag binder sequence can be bound by the streptavidin receptor.Binding of streptavidin to the Strep-tag sequence can then be detectedusing various techniques. Further examples of binder sequences includethe hexahistidine-tag (His-tag), FLAG-tag, calmodulin-binding peptide,covalent yet dissociable peptide, heavy chain of protein C tag, and thelike. Alternative (or additional) binder sequence-receptor pairs willalso fall within the scope of the present disclosure.

With continued reference to binder sequences as disclosed herein, eachbinder sequence will have a particular or defined amino acid sequence. Abinder sequence can include at least three amino acids. Example bindersequences disclosed here include between about five amino acids andabout twelve amino acids. However, binder sequences having less thanfive or more than twelve amino acids can also be used. The positions ofeach amino acid in a particular binder sequence can be defined startingat either the N-terminus ([N]) or C-terminus ([C]). For example, thepositions of the amino acids in the aforementioned Strep-tag bindersequence can be defined as [N]-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-[C] (SEQID NO:3). Accordingly, the position of the amino acid Histidine (His) isdefined as the third amino acid from the N-terminus of the Strep-tagbinder sequence. Notably, and as described above, the Strep-tag bindersequence can be flanked by one or more additional amino acids at eitheror both of the N-terminus and the C-terminus.

A population of peptide features as disclosed herein can further includeone or more control peptides or control peptide features comprisingmultiple control peptides. A variety of control peptides having variousfunctions or purposes can be included in a particular population ofpeptide features. However, at least a portion of these control peptidescan be synthesized to have an amino acid sequence including a bindersequence. In one example, a control peptide amino acid sequence consistsof the binder sequence. In another example, a control peptide amino acidsequence includes the binder sequence flanked by one or more additionalamino acids at either or both of the N-terminus and the C-terminus.Control peptide features that are correctly synthesized and thereforeinclude a binder sequence can be bound by a receptor having an affinityfor the included binder sequence. On the other hand, control peptidefeatures that are incorrectly synthesized may be bound with an alteredaffinity (or not bound at all) by the receptor. In the example case ofthe Strep-tag, a substitution or deletion including a selected one ofthe amino acids in the binder sequence (e.g., the amino acid His at thethird position from the N-terminus of the Strep-tag binder sequence) maypartially or completely disrupt the ability of the correspondingstreptavidin receptor to bind the incorrectly synthesized controlpeptide that includes the Strep-tag binder sequence.

A method according to the present disclosure further includes detectinga signal output characteristic of an interaction of the receptor with acontrol peptide feature. A step of detecting a signal output can includeany manner of monitoring or otherwise observing a measurable aspect ofone or more peptides or peptide features within a population of peptidesin the presence or absence of a receptor. Example signal outputs includean optical output (e.g., luminescence), an electrical output, a chemicaloutput, the like, and combinations thereof. As a result, the step ofdetecting the signal output can include measuring, recording, orotherwise observing the signal output using any suitable instrument.Example instruments include optical and digital detection instrumentssuch as fluorescence microscopes, digital cameras, or the like. In someembodiments, detecting a signal output further includes a perturbationsuch as excitation with light at one or more wavelengths, thermalmanipulation, introduction of one or more chemical reagents, the like,and combinations thereof.

In some embodiments of the present system and method, the detectedsignal output is characteristic of an interaction of the receptor with acontrol peptide feature. As discussed above, depending on the actualsequence of the control peptide synthesized to incorporate the bindersequence, the receptor may have a variable interaction with the controlpeptide. For an example receptor-binder sequence pair, the receptorexhibits a strong affinity for a control peptide having the correctbinder sequence; however, for a different control peptide having thebinder sequence but possessing a synthesis error (e.g., an amino acidmodification, substitution, or deletion within the binder sequence), thereceptor exhibits a relatively weaker affinity for the flawed controlpeptide. The affinity (or interaction) of the receptor for each of thecorrect and flawed control peptides may be detected as a signal outputcharacteristic of the interaction. Therefore, a corresponding signaloutput can be indicative of the fidelity of incorporation of the bindersequence into a control peptide or the quality of the control peptide,in general.

Returning again to the Strep-tag example, two distinct control peptidesare synthesized to have the Strep-tag binder sequence. One of thecontrol peptides (control peptide A) is accurately synthesized andpossesses the full length Strep-tag binder sequence. The other of thecontrol peptides (control peptide B) is synthesized incorrectly and as aresult includes a deletion of the amino acid His at the third positionfrom the N-terminus within the Strep-tag binder sequence. In the casethat the His in question contributes the affinity of the streptavidinreceptor to the Strep-tag binder sequence, the streptavidin receptorwill have a higher affinity for control peptide A as compared withcontrol peptide B. As a result, incubating each of control peptide A andcontrol peptide B with a fluorescently-labelled streptavidin receptorleads to a relatively greater concentration of the labelled streptavidinreceptor at the location of control peptide A and a relatively smallerconcentration of the labelled streptavidin receptor at the location ofcontrol peptide B. The resulting differential fluorescent signal outputfrom the locations of each of the control peptides is thereforecharacteristic of the interaction of the receptor with the controlpeptides. In particular, the signal output due to the streptavidinreceptor affinity for the Strep-tag binder sequence is indicative of thefidelity of incorporation of the amino acid His into the controlpeptide. If each of the control peptides synthesized to have theStrep-tag binder sequence are interrogated and found to bind thestreptavidin receptor more weakly than would be expected, it can beinferred that the greater population of peptides comprising the controlpeptides may also include synthesis errors related to the quality ordelivery of synthesis reagents.

With continued reference to control peptides A and B from theaforementioned Strep-tag example, it will be appreciated that staggeringthe synthesis of the control peptides relative to one another may beuseful for longitudinal analysis of the overall peptide synthesisoperation. In one example, peptide A was synthesized beginning with thefirst period of a multi-period peptide synthesis operation, whilepeptide B was synthesized beginning at a later period (i.e., synthesisof peptide B was delayed relative to peptide A). Upon determining thatthere is differential binding of the control peptides A and B by thestreptavidin receptor, it can be inferred that there was a change in thequality of peptide synthesis over the course of the overall peptidesynthesis operation. In one aspect, an accurately synthesized controlpeptide A and an inaccurately synthesized control peptide B may beindicative of degradation of one or more peptide synthesis reagents overtime, a malfunction or change to the process equipment used for peptidesynthesis, or the like. In another aspect, if both control peptides Aand B are found to include one or more synthesis errors (as determinedby streptavidin receptor binding), then errors due to a temporal orlongitudinal effect on peptide synthesis may be ruled out as a cause. Itwill be further appreciated that other patterns or observations based ontemporally or longitudinally staggered control peptide synthesis canenable assessment and diagnosis of yet other peptide synthesis outcomes.

Notably, a synthetic peptide population can include a population ofpeptide features that is synthesized to include alternative buildingblocks such as non-natural amino acids, amino acid derivatives, or othermonomer units altogether. In this case, one or more binder sequences canbe prepared with each of the selected alternative building blocks. Thebinder sequences can then be used to interrogate the fidelity ofincorporation of each of the alternative building blocks into acorresponding binder sequence. For example, it may be useful tosynthesize a population of peptide features where at least some of thepeptide features include the non-natural amino acid citrulline. In orderto monitor whether citrulline was successfully incorporated into thepopulation of peptide features, a binder sequence including at least onecitrulline within the binder sequence that contributes to receptorbinding can be identified. The binder sequence can be included as acontrol peptide feature within the overall population of peptidefeatures. Further variations and alternative methodologies for assessingthe fidelity of a synthetic peptide population according to the presentdisclosure will become apparent from the following detailed description.

II. Peptides

According to various embodiments of the instant disclosure, peptides(e.g., control peptides, peptide binder sequences) are disclosed. Eachof the peptides includes two or more natural or non-natural amino acidsas described herein. In examples described herein, a linear form ofpeptide is shown. However, one of skill in the art would immediatelyappreciate that the peptides can be converted to a cyclic form, e.g., byreacting the N-terminus with the C-terminus as disclosed in the U.S.Pat. Pub. No. 2015/0185216 to Albert et al. and filed on Dec. 19, 2014.The embodiments of the invention therefore include both cyclic peptidesand linear peptides.

As used herein, the terms “peptide,” “oligopeptide,” and “peptidebinder” refer to organic compounds composed of amino acids, which may bearranged in either a linear chain (joined together by peptide bondsbetween the carboxyl and amino groups of adjacent amino acid residues),in a cyclic form (cyclized using an internal site) or in a constrainedform (e.g., “macrocycle” of head-to-tail cyclized form). The terms“peptide” or “oligopeptide” also refer to shorter polypeptides, i.e.,organic compounds composed of less than 50 amino acid residues. Amacrocycle (or constrained peptide), as used herein, is used in itscustomary meaning for describing a cyclic small molecule such as apeptide of about 500 Daltons to about 2,000 Daltons.

The term “natural amino acid” or “canonical amino acid” refers to one ofthe twenty amino acids typically found in proteins and used for proteinbiosynthesis as well as other amino acids which can be incorporated intoproteins during translation (including pyrrolysine and selenocysteine).The twenty natural amino acids include the L-stereoisomers of histidine(His; H), alanine (Ala; A), valine (Val; V), glycine (Gly; G), leucine(Leu; L), isoleucine (Ile; I), aspartic acid (Asp; D), glutamic acid(Glu; E), serine (Ser; S), glutamine (Gln; Q), asparagine (Asn; N),threonine (Thr; T), arginine (Arg; R), proline (Pro; P), phenylalanine(Phe; F), tyrosine (Tyr; Y), tryptophan (Trp; W), cysteine (Cys; C),methionine (Met; M), and lysine (Lys; K). The term “all twenty aminoacids” refers to the twenty natural amino acids listed above.

The term “non-natural amino acid” refers to an organic compound that isnot among those encoded by the standard genetic code, or incorporatedinto proteins during translation. Therefore, non-natural amino acidsinclude amino acids or analogs of amino acids, but are not limited to,the D-stereoisomers of all twenty amino acids, the beta-amino-analogs ofall twenty amino acids, citrulline, homocitrulline, homoarginine,hydroxyproline, homoproline, ornithine, 4-amino-phenylalanine,cyclohexylalanine, α-aminoisobutyric acid, N-methyl-alanine,N-methyl-glycine, norleucine, N-methyl-glutamic acid, tert-butylglycine,α-aminobutyric acid, tert-butylalanine, 2-aminoisobutyric acid,α-aminoisobutyric acid, 2-aminoindane-2-carb oxylic acid,selenomethionine, dehydroalanine, lanthionine, γ-amino butyric acid, andderivatives thereof wherein the amine nitrogen has been mono- ordi-alkylated.

According to embodiments of the instant disclosure, peptides arepresented immobilized on a support surface (e.g., a microarray, a bead,or the like). In some embodiments, peptides selected for use as controlpeptides may optionally undergo one or more rounds of extension andmaturation processes to yield the control peptides disclosed herein.

III. Microarrays

The control peptides disclosed herein can be generated usingoligopeptide microarrays. As used herein, the term “microarray” refersto a two dimensional arrangement of features on the surface of a solidor semi-solid support. A single microarray or, in some cases, multiplemicroarrays (e.g., 3, 4, 5, or more microarrays) can be located on onesolid support. For a solid support having fixed dimensions, the size ofthe microarrays depends on the number of microarrays on the solidsupport. That is, the higher the number of microarrays per solidsupport, the smaller the arrays have to be to fit on the solid support.The arrays can be designed in any shape, but preferably they aredesigned as squares or rectangles. The ready to use product is theoligopeptide microarray on the solid or semi-solid support (microarrayslide).

The terms “peptide microarray” or “oligopeptide microarray,” or “peptidechip,” or “peptide epitope microarray” refer to a population orcollection of peptides displayed on a microarray, i.e., a solid surface,for example a glass, carbon composite or plastic array, slide, or chip.

The term “feature” refers to a defined area on the surface of amicroarray. The feature comprises biomolecules, such as peptides (i.e.,a peptide feature), nucleic acids, carbohydrates, and the like. Onefeature can contain biomolecules with different properties, such asdifferent sequences or orientations, as compared to other features. Thesize of a feature is determined by two factors: i) the number offeatures on an array, the higher the number of features on an array, thesmaller is each single feature, ii) the number of individuallyaddressable aluminum mirror elements which are used for the irradiationof one feature. The higher the number of mirror elements used for theirradiation of one feature, the bigger is each single feature. Thenumber of features on an array may be limited by the number of mirrorelements (pixels) present in the micromirror device. For example, thestate of the art micromirror device from Texas Instruments, Inc.(Dallas, Tex.) currently contains 4.2 million mirror elements (pixels),thus the number of features within such exemplary microarray istherefore limited by this number. However, higher density arrays arepossible with other micromirror devices.

The term “solid or semi-solid support” refers to any solid material,having a surface area to which organic molecules can be attached throughbond formation or absorbed through electronic or static interactionssuch as covalent bonds or complex formation through a specificfunctional group. The support can be a combination of materials such asplastic on glass, carbon on glass, and the like. The functional surfacecan be simple organic molecules but can also comprise of co-polymers,dendrimers, molecular brushes, and the like.

The term “plastic” refers to synthetic materials, such as homo- orhetero-co-polymers of organic building blocks (monomer) with afunctionalized surface such that organic molecules can be attachedthrough covalent bond formation or absorbed through electronic or staticinteractions such as through bond formation through a functional group.Preferably the term “plastic” refers to polyolefin, which is a polymerderived by polymerization of an olefin (e.g., ethylene propylene dienemonomer polymer, polyisobutylene). Most preferably, the plastic is apolyolefin with defined optical properties, like TOPAS® or ZEONOR/EX®.

The term “functional group” refers to any of numerous combinations ofatoms that form parts of chemical molecules, that undergo characteristicreactions themselves, and that influence the reactivity of the remainderof the molecule. Typical functional groups include, but are not limitedto, hydroxyl, carboxyl, aldehyde, carbonyl, amino, azide, alkynyl,thiol, and nitril. Potentially reactive functional groups include, forexample, amines, carboxylic acids, alcohols, double bonds, and the like.Preferred functional groups are potentially reactive functional groupsof amino acids such as amino groups or carboxyl groups.

Various methods for the production of oligopeptide microarrays are knownin the art. For example, spotting prefabricated peptides or in situsynthesis by spotting reagents (e.g., on membranes) exemplify knownmethods. Other known methods used for generating peptide arrays ofhigher density are the so-called photolithographic techniques, where thesynthetic design of the desired biopolymers is controlled by suitablephotolabile protecting groups (PLPG) releasing the linkage site for therespective next component (amino acid, oligonucleotide) upon exposure toelectromagnetic radiation, such as light (Fodor et al., (1993) Nature364:555-556; Fodor et al., (1991) Science 251:767-773). Two differentphotolithographic techniques are known in the state of the art. Thefirst is a photolithographic mask, used to direct light to specificareas of the synthesis surface effecting localized deprotection of thePLPG. “Masked” methods include the synthesis of polymers utilizing amount (e.g., a “mask”) which engages a substrate and provides a reactorspace between the substrate and the mount. Exemplary embodiments of such“masked” array synthesis are described in, for example, U.S. Pat. Nos.5,143,854 and 5,445,934, the disclosures of which are herebyincorporated by reference. Potential drawbacks of this technique,however, include the need for a large number of masking steps resultingin a relatively low overall yield and high costs, e.g., the synthesis ofa peptide of only six amino acids in length could require over 100masks. The second photolithographic technique is the so-called masklessphotolithography, where light is directed to specific areas of thesynthesis surface effecting localized deprotection of the PLPG bydigital projection technologies, such as micromirror devices(Singh-Gasson et al., Nature Biotechn. 17 (1999) 974-978). Such“maskless” array synthesis thus eliminates the need for time-consumingand expensive production of exposure masks. It should be understood thatthe embodiments of the systems and methods disclosed herein may compriseor utilize any of the various array synthesis techniques describedabove.

The use of PLPG (photolabile protecting groups), providing the basis forthe photolithography based synthesis of oligopeptide microarrays, iswell known in the art. Commonly used PLPG for photolithography basedbiopolymer synthesis are for exampleα-methyl-6-nitropiperonyl-oxycarbonyl (MeNPOC) (Pease et al., Proc.Natl. Acad. Sci. USA (1994) 91:5022-5026),2-(2-nitrophenyl)-propoxycarbonyl (NPPOC) (Hasan et al. (1997)Tetrahedron 53: 4247-4264), nitroveratryloxycarbonyl (NVOC) (Fodor etal. (1991) Science 251:767-773) and 2-nitrobenzyloxycarbonyl (NBOC).

Amino acids have been introduced in photolithographic solid-phasepeptide synthesis of oligopeptide microarrays, which were protected withNPPOC as a photolabile amino protecting group, wherein glass slides wereused as a support (U.S. App. Pub. No. 20050101763). The method usingNPPOC protected amino acids has the disadvantage that the half-life uponirradiation with light of all (except one) protected amino acids iswithin the range of approximately 2 to 3 minutes under certainconditions. In contrast, under the same conditions, NPPOC-protectedtyrosine exhibits a half-life of almost 10 minutes. As the velocity ofthe whole synthesis process depends on the slowest sub-process, thisphenomenon increases the time of the synthesis process by a factor of 3to 4. Concomitantly, the degree of damage by photogenerated radical ionsto the growing oligomers increases with increasing and excessive lightdose requirement.

As understood by one of skill in the art, peptide microarrays comprisean assay principle whereby thousands (or in the case of the instantdisclosure, millions) of peptides (in some embodiments presented inmultiple copies) are linked or immobilized to the surface of a solidsupport (which in some embodiments comprises a glass, carbon compositeor plastic chip or slide).

In some embodiments, a peptide microarray is exposed to a sample ofinterest such as a receptor, antibody, enzyme, peptide, oligonucleotide,or the like. The peptide microarray exposed to the sample of interestundergoes one or more washing steps, and then is subjected to adetection process. In some embodiments, the array is exposed to anantibody targeting the sample of interest (e.g. anti-IgG human/mouse oranti-phosphotyrosine or anti-myc). Usually, the secondary antibody istagged by a fluorescent label that can be detected by a fluorescencescanner. Other detection methods are chemiluminescence, colorimetry, orautoradiography. In other embodiments, the sample of interest isbiotinylated, and then detected by streptavidin conjugated to afluorophore. In yet other embodiments, the protein of interest is taggedwith specific tags, such as His-tag, FLAG-tag, Myc-tag, etc., anddetected with a fluorophore-conjugated antibody specific for the tag.

After scanning the microarray slides, the scanner records a 20-bit,16-bit or 8-bit numeric image in tagged image file format (*.tif). Thetif-image enables interpretation and quantification of each fluorescentspot on the scanned microarray slide. This quantitative data is thebasis for performing statistical analysis on measured binding events orpeptide modifications on the microarray slide. For evaluation andinterpretation of detected signals an allocation of the peptide spot(visible in the image) and the corresponding peptide sequence has to beperformed.

A peptide microarray is a slide with peptides spotted onto it orassembled directly on the surface by in situ synthesis. Peptides areideally covalently linked through a chemoselective bond leading topeptides with the same orientation for interaction profiling.Alternative procedures include unspecific covalent binding and adhesiveimmobilization.

According one specific embodiment of the instant disclosure, thespecific peptide binders are identified using maskless array synthesisin the fabrication of the peptide binder probes on the substrate.According to such embodiments, the maskless array synthesis employedallows ultra-high density peptide synthesis of up to 2.9 million uniquepeptides, with each of the 2.9 million features/regions having up to 10⁷reactive sites that could yield a full-length peptide. Smaller arrayscan also be designed. For example, an array representing a comprehensivelist of all possible 5-mer peptides using 19 natural amino acidsexcluding cysteine will have 2,476,099 peptides. In other examples, anarray may include non-natural amino acids as well as natural aminoacids. An array of 5-mer peptides by using all combinations of 18natural amino acids excluding cysteine and methionine may also be used.Additionally, an array can exclude other amino acids or amino aciddimers. In some embodiments, an array may be designed to exclude anydimer or a longer repeat of the same amino acid, as well as any peptidecontaining HR, RH, HK, KH, RK, KR, HP, and PQ sequences to create alibrary of 1,360,732 unique peptides. Smaller arrays may have replicatesof each peptide on the same array to increase the confidence of theconclusions drawn from array data.

In various embodiments, the peptide arrays described herein can have atleast 1.6×10⁵ peptides, or up to about 1.0×10⁸ peptides or any numberin-between, attached to the solid support of the peptide array. Asdescribed herein, a peptide array comprising a particular number ofpeptides can mean a single peptide array on a single solid support, orthe peptides can be divided and attached to more than one solid supportto obtain the number of peptides described herein.

Arrays synthesized in accordance with such embodiments can be designedfor peptide binder discovery in the linear or cyclic form (as notedherein) and with and without modification such as N-methyl or otherpost-translational modifications. Arrays can also be designed forfurther extension of potential binders using a block-approach byperforming iterative screens on the N-terminus and C-terminus of apotential hit (as is further described in detail herein). Once a hit ofan ideal affinity has been discovered it can be further matured using acombination of maturation arrays (described further herein), that allowa combinatorial insertion, deletion and replacement analysis of variousamino acids both natural and non-natural.

The peptide arrays of the instant disclosure are used to identify thespecific binders or binder sequences of the invention as well as formaturation and extension of the binder sequences for use in the designand selection of control peptides.

IV. Design and Synthesis of Control Peptide Features

Turning now to FIG. 1, one embodiment of the present disclosure providesfor a peptide array with a portion 100 of the peptide array including aplurality of control peptide features 102 for longitudinal analysis ofthe quality of a broader population of peptide features (not shown)including the control peptide features 102. While only a portion 100 ofthe peptide array is shown in FIG. 1, it will be appreciated that thepeptide array can be designed comprising a population of hundreds,thousands, tens of thousands, hundreds of thousands and even millions ofpeptide features including or in addition to the control peptidefeatures 102. In some embodiments, the population of peptide features onthe portion 100 of the peptide array can be configured such that thepeptide features collectively represent an entire protein, gene,chromosome, or even an entire genome of interest (e.g., a humanproteome). Additionally, the peptide features can be configuredaccording to specific criteria, whereby specific amino acids or motifsare excluded. Furthermore, the peptide features can be configured suchthat each of the peptide sequences comprises an identical length. Forexample, in some embodiments, the population of peptide featuresimmobilized on an array substrate may all comprise 3-, 4-, 5-, 6-, 7-,8-, 9-, 10-, 11-, or even 12-mers, or more. Notably, the sequences ofthe peptide features at specific locations on the array are known.

With reference to the portion 100 of the peptide array of FIG. 1, thecontrol peptide features 102 can be repeated at a plurality of locationson the peptide array. The peptide array is illustrated to includenineteen control peptide features 102 within the portion 100. However,the peptide array can include a number of additional control peptidefeatures 102. Alternative embodiments of peptide arrays according to thepresent disclosure can include any suitable number of control peptidefeatures based on factors such as the size of the peptide array (e.g.,number of total peptide features, number of features per unit area), thefidelity of the synthesis process, the number and type of reagents used(e.g., natural amino acids, non-natural amino acids, non-amino acidreagents), statistical requirements (e.g., number of replicates,statistical methods relied on), and the like. It will be appreciatedthat, while the Figures illustrate the use of control peptide featuressynthesized to have the same sequence, embodiments of the presentdisclosure include peptide arrays having two or more control peptidesequences. Moreover, the two or more different control peptide sequencescan be bound by the same receptor or different receptors.

In the embodiment illustrated in FIG. 1, each of the control peptidefeatures 102 within the portion 100 are composed of a plurality ofpeptides that share the common peptide binder sequence [N]-WTHPQFE-[C](i.e., WTHPQFE (SEQ ID NO:1)) that is selectively bound by the receptorstreptavidin ([N] and [C] designate the N-terminus and C-terminus of thebinder sequence, respectively). More particularly, each one of thecontrol peptide features 102 is synthesized to be identical to eachother one of the control peptide features 102 upon completion ofsynthesis of all the broader population of peptide features on thepeptide array 100. Accordingly, if each of the nineteen control peptidefeatures 102 in FIG. 1 is successfully synthesized without theoccurrence of one or more substitutions, insertions, deletions, otherlike errors, or combinations thereof, then the control peptide features102 will comprise a plurality of identical peptides, with each peptidehaving the peptide binder sequence WTHPQFE (SEQ ID NO:1).

In one aspect, while each of the control peptide features 102 may beidentical following completion of synthesis, the timing of synthesis foreach of the control peptide feature 102 can vary. As illustrated in FIG.1, a first subset 104 of the control peptide features 102 (indicated assquare features with diagonal hatching) are synthesized beginning with afirst synthesis period of a twelve synthesis period process. Eachsynthesis period is divided into a series of cycles where a single aminoacid is exposed to the peptide array 100 per cycle for the step-wiseassembly of the population of peptide features. For example, a firstcycle can correspond to the addition of alanine, a second cycle to theaddition of arginine, a third cycle to the addition of asparagine, andso forth. In the present example, each synthesis period is divided into20 cycles, with each cycle corresponding to one of the twenty canonicalamino acids as shown in Table 1. Over the course of N consecutivesynthesis periods, a peptide array will be exposed to each amino acid(or other building block) N times, where N is a positive integer.Accordingly, in the present synthesis example having twelve periods, thepeptide array in FIG. 1 is exposed to each amino acid a total of twelvetimes.

TABLE 1 Amino Acid Amino Acid Cycle (3-letter code) (1-letter code) 1Ala A 2 Arg R 3 Asn N 4 Asp D 5 Cys C 6 Gln E 7 Glu Q 8 Gly G 9 His H 10Ile I 11 Leu L 12 Lys K 13 Met M 14 Phe F 15 Pro P 16 Ser S 17 Thr T 18Trp W 19 Tyr Y 20 Val V

In the present example, the peptide binder sequence WTHPQFE (SEQ IDNO:1) can be synthesized from the C-terminus to the N-terminus over aminimum of three synthesis periods. With reference to Table 1, the aminoacids Gln and Phe are synthesized in a first synthesis periods followedby the amino acids Glu and Pro in a second synthesis period and endingwith the amino acids His, Thr, and Trp in a third synthesis period. Foran example synthesis process having twelve synthesis periods, it ispossible to delay initiation of synthesis of a control peptide havingthe peptide binder sequence WTHPQFE (SEQ ID NO:1) by up to and includingnine periods, with synthesis beginning in the tenth period and takingplace over synthesis periods ten, eleven, and twelve. In the designillustrated in FIG. 1, synthesis of at least one of the control peptidefeatures 102 is initiated beginning with each one of the first ten (ofthe twelve total) synthesis periods. The first subset 104 of the controlpeptide feature 102 is synthesized beginning with the first one of thesynthesis periods. Synthesis of the first subset 104 is then completedover the next two sequential synthesis periods (i.e., the second andthird synthesis periods). A second subset 106 of the control peptidefeatures 102 is synthesized beginning with the second synthesis period(i.e., after the first synthesis period). Synthesis of the second subset106 is then completed over the next two sequential synthesis periods(i.e., the third and fourth synthesis periods). As a result, synthesisof the second subset 106 is delayed by one synthesis period relative tothe first subset 104, and also overlaps with synthesis of the firstsubset 104 during the second and third synthesis periods.

The control peptide features 102 include additional subsets of controlpeptide features as illustrated in FIG. 1 such that synthesis of atleast one of the control peptide features 102 begins in each of thefirst ten of the twelve total synthesis periods. In one aspect, thecontrol peptide features 102 include a third subset 108, a fourth subset110, a fifth subset 112, a sixth subset 114, and seventh subset 116, aneighth subset 118, a ninth subset 120, and a tenth subset 122. Incomparison with the first subset 104 and second subset 106, the subsets110-122 are each delayed by one synthesis period relative to theprevious subset of the control peptide features 102. For example,initiation of synthesis of the third subset 108 begins with the thirdsynthesis period, which is delayed by one synthesis period relative toinitiation of synthesis of the second subset 106 (which begins with thesecond synthesis period). By extension, synthesis of the tenth subset122, which is delayed by one synthesis period relative to the ninthsubset 120, begins with the tenth period and is completed over theeleventh and twelfth synthesis periods.

Turning now to FIGS. 2 and 3, another example peptide array 200 includesa plurality of control peptide features 202 immobilized on an arraysubstrate 204 that includes a solid support 206 having a reactivesurface 208 (e.g., a reactive amine layer). Each of the peptidesequences of the control peptides features 202 is based on thestreptavidin peptide binder sequence WTHPQFE (SEQ ID NO:1). Furthermore,while each of the control peptide features 202 is illustrated as asingle peptide, it will be appreciated that each of the control peptidefeatures 202 includes a plurality of co-localized peptides sharing thesame amino acid sequence. Notably, there may be variations in the actualsequences of each of the peptides within a given peptide feature due tovarious limitations associated with synthesis process. However, for thepurposes of illustration, the peptides within a peptide feature areassumed to comprise the same sequence.

The control peptide features 202 are synthesized over the course of ninesynthesis periods, where each synthesis period is further divided intotwenty synthesis cycles as shown in FIG. 3. The streptavidin peptidebinder sequence WTHPQFE (SEQ ID NO:1), which is synthesized from theC-terminus to the N-terminus, can be synthesized over a minimum of threeperiods based on the order of the synthesis cycles for a synthesisperiod as shown in the table. According to one approach of the presentdisclosure, the control peptide features 202 are each built over threeconsecutive synthesis periods without skipping synthesis cycles orsynthesis periods. For example, the amino acids Gln and Phe aresynthesized in a first synthesis period followed by the amino acids Gluand Pro in a second synthesis period and ending with the amino acidsHis, Thr, and Trp in a third synthesis period, where each of the first,second, and third synthesis periods are uninterrupted consecutivesynthesis periods. Using this uninterrupted synthesis approach, a firstsubset 210 of the control peptide features 202 is synthesized beginningwith a first (initial) synthesis period, a second subset 212 of thecontrol peptide features 202 is synthesized beginning with a secondsynthesis period, and a third subset 214 of the control peptide features202 is synthesized beginning with a third synthesis period. Similarly, afourth subset 216 of the control peptide features 202 is synthesizedbeginning with a fourth synthesis period, a fifth subset 218 of thecontrol peptide features 202 is synthesized beginning with a fifthsynthesis period, and so forth, where consecutively numbered synthesisperiods are carried out in an uninterrupted consecutive manner.

Given that each of the control peptide features 202 is synthesized in anuninterrupted manner, it can be seen from FIG. 2 that only the firstfour synthesis periods have been completed (at least through cycleeighteen of synthesis period four; see Table 1) as the first subset 210and the second subset 212 are illustrated as having the full lengthstreptavidin binder sequence. The third subset 214 and fourth subset 216are only partially complete, and synthesis of the fifth subset 218 hasnot yet begun, which indicates that the peptide synthesis operation iscurrently shown for a synthesis state between synthesis cycle nineteenof synthesis period four and synthesis cycle five of synthesis periodfive. In summary, the intermediate synthesis state in FIG. 2 illustratesthat synthesis of the control peptide features 202 is staggered with aportion of the control peptide features 202 synthesized beginning withthe initial or first synthesis period, and another portion of thecontrol peptide features 202 synthesized beginning with subsequentsynthesis periods (i.e., synthesis is delayed relative to the firstsynthesis period). As a result, the control peptide features 202 arecontinuously synthesized across each of the synthesis periods in a givensynthesis operation.

With reference to synthesis scheme illustrated in FIG. 3, uninterruptedsynthesis of the streptavidin peptide binder sequence WTHPQFE (SEQ IDNO:1) starting from the C-terminus is achieved over three synthesisperiods given the illustrated order of synthesis cycles (see also Table1). A first subset of control peptide features is synthesized oversynthesis periods one, two, and three, with each of the relevantsynthesis cycles used indicated by dashed squares. Following along withsynthesis of the first subset of control peptide features, the aminoacids Gln and Phe are synthesized in the first synthesis period atsynthesis cycles six and fourteen, respectively. Next, the amino acidsGlu and Pro are synthesized in the second synthesis period at synthesiscycles seven and fifteen, respectively. Finally, the amino acids His,Thr, and Trp are synthesized in the third synthesis period duringcorresponding synthesis cycles nine, seventeen, and eighteen. Notably,synthesis of other subsets of control peptide features is delayedrelative the first subset of control peptide features. In one aspect, asecond subset of control peptide features is synthesized over synthesisperiods two, three, and four as indicated by solid squares. In anotheraspect, a sixth subset of control peptide features is synthesized oversynthesis periods six, seven, and eight as indicated by dashed circles.In yet another aspect, a seventh subset of control peptide features issynthesized over synthesis periods seven, eight, and nine as indicatedby solid circles.

Whereas synthesis examples are explicitly shown for first, second,sixth, and seventh subsets of control peptide features, other subsets ofcontrol peptide features can be synthesized over the course of thesynthesis operation illustrated in FIG. 3. For example, a third subsetof control peptide features can be synthesized over synthesis periodsthree, four, and five such that synthesis of the third subset of controlpeptide features is delayed by one synthesis period relative to thesecond subset (solid squares). Moreover, the third subset of controlpeptide features is delayed by two synthesis periods relative to thefirst subset (dashed squares). Notably, control peptide features can besynthesized beginning with any of synthesis periods one through seven inorder to complete synthesis of the control peptide features by the endof synthesis period nine (see, for example, the seventh subset ofcontrol peptide features indicated by solid circles).

According to another embodiment of the present disclosure, synthesis ofone or more control peptide features is distributed across a greaternumber of synthesis periods or synthesis cycles than the minimum numberrequired. For example, control peptide features can be synthesizedacross three or more consecutive or nonconsecutive synthesis periods. Asdescribed above and with reference to FIG. 3, synthesis of thestreptavidin peptide binder sequence WTHPQFE (SEQ ID NO:1) may beachieved over a minimum of three synthesis periods. Turning to FIG. 4,the same streptavidin peptide binder sequence can be built in a numberof alternative synthesis patterns over three or more consecutive ornon-consecutive synthesis periods. In one example scheme, a first subsetof control peptide features is synthesized over synthesis periods two,three, four, seven, and eight with each of the relevant synthesis cyclesused indicated by solids stars. Following along with synthesis of thefirst subset of control peptide features, the amino acids Gln and Pheare synthesized starting in the second synthesis period (i.e., skippingthe first synthesis period altogether) at synthesis cycles six andfourteen, respectively. Next, the amino acid Glu is synthesized in thethird synthesis period at synthesis cycle seven. Then, the amino acidPro is synthesized in the fourth synthesis period at synthesis cyclefifteen. After skipping synthesis periods five and six, the amino acidHis is synthesized in the seventh synthesis period at synthesis cyclenine. Finally, the amino acids Thr and Trp are synthesized in the eighthsynthesis period during corresponding synthesis cycles seventeen andeighteen.

With continued reference to FIG. 4, synthesis of other subsets ofcontrol peptide features is both delayed relative to, and overlappingwith, the first subset of control peptide features. In one aspect, asecond subset of control peptide features is synthesized in a continuousand uninterrupted manner over synthesis periods two, three, and four asindicated by solid squares. In another aspect, a third subset of controlpeptide features is synthesized in a semi-continuous manner oversynthesis periods three, four, five, six, seven, eight, and nine asindicated by dashed circles. The term semi-continuous with respect tosynthesis of the third subset indicates that the one or more possiblesynthesis cycles is skipped for a given synthesis period. For example,each of the amino acids Gln and Phe may be synthesized sequentiallywithin the same synthesis period to completely synthesize thestreptavidin binder sequence in the minimum possible number of synthesisperiods and synthesis cycles. However, synthesis of the amino acids Glnand Phe is distributed across two different synthesis periods (i.e.,synthesis periods three and four) for the third subset. Accordingly,although synthesis of the third subset is carried out over sevenconsecutive synthesis periods with no intermediate synthesis periodsskipped or otherwise omitted between synthesis periods three and nine,synthesis of the third step is considered only semi-continuous asseveral possible synthesis cycles are skipped. More generally, asequence or feature synthesized over a number of consecutive synthesisperiods that is greater than the minimum possible number of synthesisperiods is considered to have been synthesized semi-continuously.

Synthesis of the first subset, second subset, and third subset ofcontrol peptides illustrated in FIG. 4 overlaps in a number of ways. Inone aspect, synthesis of the first subset overlaps with synthesis ofsecond subset at synthesis cycle fifteen of synthesis period four.Synthesis of the first subset also overlaps with synthesis of thirdsubset at synthesis cycle seventeen of synthesis period eight. Inanother aspect, synthesis of the second subset overlaps with synthesisof third subset at synthesis cycle six of synthesis period three.Accordingly, synthesis of each pair of the three subsets of controlpeptides in FIG. 4 overlaps for at least one particular combination of asynthesis period and synthesis cycle. Further synthesis overlaps canalso be seen for different subsets of control peptides that share aparticular synthesis period but not necessarily a synthesis cycle withinthe particular synthesis period. In one example, each of the firstsubset, second subset, and third subset of control peptides arepartially synthesized during both the third and fourth synthesisperiods. In another example, each of the second subset and third subset(but not the first subset) of control peptides are partially synthesizedduring the fifth synthesis period.

Notably, many variations and schemes are possible for the design of howand where synthesis of the various subsets of control peptide featuresoverlaps. In some embodiments, it may be useful to have a design similarto that shown in FIG. 3 where a subset of control peptide features isinitiated in each of the synthesis periods with each subset of controlpeptide features being further synthesized in a continuous anduninterrupted manner. In other embodiments, it may be useful to have adesign similar to that shown in FIG. 4 where synthesis of at least aportion of the various subsets of control peptide features is carriedout in a semi-continuous or discontinuous, interrupted manner. Each ofthe subsets of control peptide features can be synthesized using thesame patterns of synthesis periods and synthesis cycles but delayinginitiation of synthesis of each subset of control peptide features byone or more synthesis periods relative to another one or more of thecontrol peptide features. Alternatively, each of the subsets of controlpeptide features can be synthesized using different synthesis patterns(e.g., as shown in FIG. 4).

In some embodiments, the design of synthesis for the control peptidefeatures can be leveraged to generate an output that can be used tointerpret whether or not one or more synthesis errors occurred during agiven peptide synthesis operation. Turning to FIGS. 5-7, an example oflongitudinal assessment of a synthetic peptide population can generallyinclude interrogating a set of control peptide features synthesized overa plurality of synthesis periods in the presence of a receptor having anaffinity for a binder sequence included in the control peptide features,and detecting a signal output characteristic of the interaction of thereceptor with the control peptide features.

With reference to FIG. 5, a peptide array 300 includes a population ofcontrol peptide features 302 immobilized on an array substrate 304 andsynthesized over the course of several synthesis periods. Each of thecontrol peptide features 302 includes a plurality of colocalizedpeptides sharing the same amino acid sequence. It will be appreciatedthat in actuality, there may be variations in the actual sequences ofeach of the peptides within a given peptide feature due to variouslimitations associated with the synthesis process. However, for thepurposes of illustration, the peptides within a peptide feature areassumed to comprise the same sequence. Depending on the synthesis methodemployed, a peptide feature may have a varying footprint or featuredensity. In one example, a peptide feature has a footprint of about 10μm×10 μm square and includes up to about 10⁷ individual peptides.However, other footprints and feature densities are possible as will berecognized by a person of ordinary skill in the art. In the presentexample, the control peptide features 302 include a plurality ofpeptides that each have the amino acid sequence of the streptavidinbinder sequence WTHPQFE (SEQ ID NO:1)).

The control peptide features 302 include a first subset 306 of controlpeptide features including a first feature 306 a, a second feature 306b, and a third feature 306 c. The control peptide features 302 furtherinclude a second subset 308 and a third subset 310 of control peptidesfeatures. Each of the control peptide features 302 are synthesized in acontinuous, uninterrupted manner as described above with respect toFIGS. 2 and 3. Accordingly, synthesis of each of the control peptidefeatures 302 is carried out over the course of three consecutivesynthesis periods. For example, the first subset 306 is synthesizedbeginning with a first one of the plurality of synthesis periods asindicated by the ‘[1]’ beneath each of the first feature 306 a, thesecond feature 306 b, and the third feature 306 c. Synthesis of thefirst subset 306 is then completed over the course of the first, second,and third synthesis periods in a continuous and uninterrupted manner.

In another aspect, the second subset 308 and the third subset 310 aresynthesized beginning after the first one of the plurality of synthesisperiods such that synthesis of the second subset 308 and the thirdsubset 310 of the control peptide features 302 is delayed by at leastone synthesis period relative to the first subset 306. In the presentexample, the second subset 308 is synthesized beginning with the secondone of the plurality of synthesis periods as indicated by the ‘[2]’beneath the illustrated feature of the second subset 308. The thirdsubset 310 is synthesized beginning with the third one of the pluralityof synthesis periods as indicated by the ‘[3]’ beneath the illustratedfeature of the third subset 310. As a result, synthesis of the firstsubset 306 overlaps with synthesis of the second subset 308 duringsynthesis periods two and three. Further, synthesis of the first subset306 overlaps with synthesis of the third subset 310 during (only)synthesis period three. Notably, the peptide array 300 can includenumerous peptide features beyond the number of features shown in theembodiment illustrated in FIGS. 5-7. Moreover, alternative or additionalpeptide features can also be included in a peptide array according tothe present disclosure.

Once the peptide array 300 has been synthesized as illustrated in FIG.5, a plurality of receptor molecules known to interact with the selectedpeptide binder sequences can be contacted to the peptide array 300 inorder to interrogate the population of control peptide features 302 inthe presence of the receptor molecules (FIG. 6). A number of receptormolecules 312 are shown as interacting with each of the feature 306 aand the feature 306 c of the first subset 306, as well as theillustrated feature of the third subset 310. Interaction of the receptormolecules 312 with the one or more of the control peptide features 302can include binding, catalysis of (or participation in) a reactionincluding peptides within the corresponding ones of the control peptidefeatures 302, digestion of the peptides within the control peptidefeatures 302, the like, and combinations thereof. In the presentexample, the receptor molecules 312 represent streptavidin moleculesused in the identification of the peptide binder sequence represented bythe peptides in the control peptide features 302 (i.e., WTHPQFE (SEQ IDNO:1)). Accordingly, a strong degree of interaction between the peptidesin the particular control peptide features 302 and the receptormolecules 312 would be anticipated as represented by the plurality ofreceptor molecules 312 associated with the various control peptidefeatures 302. In one aspect, the interaction of the receptor molecules312 with the control peptide features 302 on the peptide array 300 canbe detected, for example, by labeling the receptor molecules 312 with adetectable tag 314 or other like reporter (FIG. 7). As shown in theillustrated embodiment, the detectable tag 314 is a labeled antibodythat is specific for targeting the receptor molecules 312. However,other detection schemes are within the scope of the present disclosure.

Whereas a relatively greater number of receptor molecules 312 areassociated with the feature 306 a, the feature 306 c, and theillustrated feature of the third subset 310 in FIG. 6, relatively feweror no receptor molecules 312 are associated with any one of the feature306 b, or the illustrated feature of the second subset 308. In oneaspect, an error in synthesis during one or more of the first, second,and third synthesis periods can affect the fidelity with which thestreptavidin binder sequence is produced, thereby resulting in little tono interaction of the receptor molecules 312 with the indicated controlpeptide features 302. Similarly, the degree of interaction or therelative change in the extent of interaction of the receptor molecules312 with any of the control peptide features 302 on the peptide array300 can be interrogated. The results of the interrogation can be used toidentify during which synthesis periods or synthesis cycles an error orother deviation in peptide synthesis occurred.

One possible result determined from interrogating the interaction of thereceptor molecules 312 with the control peptide features 302 on thepeptide array 300 can include the identification of an error associatedwith a particular synthesis cycle or synthesis period. For example,FIGS. 5-7 illustrate a hypothetical synthesis run in which an erroroccurred at synthesis cycle seven (addition of the amino acid Glu)during synthesis period three. The error resulted in the improperincorporation of the amino acid Glu into the second subset 308 of thecontrol peptide features 302, leaving the corresponding features of thesecond subset 308 incapable of binding the receptor molecules 312. Thedetected signal output characteristic of the interaction of the receptormolecules 312 with the plurality of control peptide features 302indicates that the second subset 308 of features was not bound byreceptor molecules 312. By contrast, the detected signal output isindicative of binding of a relatively larger number of receptormolecules 312 to the first feature 306 a, the third feature 306 c, andthe illustrated feature of the third subset 310. Based on thisinformation, an inference can be made that an error occurred affectingonly a particular synthesis cycle as opposed to an entire synthesisperiod. Moreover, the error affecting the particular synthesis cycle waslikely confined to only one of the plurality of synthesis periods asopposed to each of the synthesis periods as the subset of the controlpeptide features 302 assembled in synthesis periods overlapping with theaffected second subset 308 of features were not affected in the sameway.

Based on the characterization of binding of the receptor molecules 312in FIG. 7, determining which synthesis cycle (or cycles) were affectedmay require additional information or analysis. In one aspect, thesignal output for features in the second subset 308 may becharacteristic of a particular amino acid deletion. For example, thepeptide array 300 may include one or more additional control peptidefeatures (not shown) that represent each possible single amino aciddeletion sequence corresponding to the streptavidin binder sequencerepresented by the control peptide features 302. The signal output forthe second subset 308 can then be compared with the signal output forthe additional control peptide features to determine whether a deletionmay have occurred, and what the likely identity of the deleted aminoacid was. If the outcome of the analysis indicates that a deletionoccurred and the identity of the deleted amino acid can be elucidated,then the corresponding synthesis period and synthesis cycle during whichthe deletion error occurred can be determined. For example, followinginterrogation of the control peptide features 302, a binding profile forthe illustrated feature of the second subset is determined to uniquelymatch with a binding profile for a control peptide binder with a knownGlu deletion synthesized on the same peptide array 300. The amino acidGlu is incorporated during the third synthesis period for the secondsubset 308. Further, the first subset 306 and third subset 310 are foundto have binding profiles that are indicative of synthesis of peptideshaving the correct, full-length binder sequence. Therefore, thedetermination can be made that only the third synthesis period wasaffected (out of at least synthesis periods two, three, and four), andfurther, that only the seventh synthesis cycle corresponding to theaddition of Glu was affected during the third synthesis period.

Another possible result determined from interrogating the peptide array300 of FIGS. 5-7 can include the identification of an error associatedwith a particular location on the array. For example, FIGS. 5-7illustrate a hypothetical synthesis run in which an error occurred forthe second feature 306 b within the first subset 306. Each of theillustrated features of the first subset 306 were synthesized inparallel (i.e., in an identical manner using the same synthesis periodsand synthesis cycles) with the only difference being the geographiclocation of synthesis on the surface of the peptide array 300. However,the second feature 306 b exhibits relatively fewer interactions with thereceptor molecules 312 as compared with either of the first feature 306a or the third feature 306 c. Accordingly, an inference can be made thatan error occurred during synthesis that affected at least the locationof the second feature 306 b. Based on the results of interrogating anyadditional feature adjacent to (or otherwise near) the second feature306 b, it may be possible to determine the quality or fidelity ofpeptide features near the second feature 306 b. Yet other informationrelated to the quality or fidelity of synthesis of the control peptidefeatures 302, and by extension the broader population of peptidefeatures can be determined based on the results of interrogating thecontrol peptide features 302 as described herein. Moreover, whereasstreptavidin-based receptor molecules and binder sequences areillustrated in the examples shown in the Figures, alternative oradditional receptor molecules and control peptide sequences can be used.Further methods and example peptide binder-receptors combinations aredescribed by Albert et al. (U.S. Pat. App. No. 2015/0185216 to Albert etal. and U.S. Prov. Pat. App. Ser. No. 62/150,202 to Albert et al.).

V. Interrogation and Detection of Longitudinal Synthesis Fidelity

Notably, the examples described with respect to FIGS. 5-7 illustrate theidentification of synthesis errors confined to particular locations,synthesis periods, synthesis cycles, or a combination thereof. However,embodiments of the present disclosure are also suited to thelongitudinal analysis of peptide synthesis. Turning to FIGS. 8-11,hypothetical signal output profiles are illustrated for two differentexample peptide arrays. A portion of a first example peptide array 400(FIGS. 8 and 9) includes a plurality of control peptide features 402synthesized over a plurality of synthesis periods in a continuousuninterrupted pattern as described previously (e.g., with respect toFIG. 3). A first subset 404 of the control peptide features 402 weresynthesized at every other location across the illustrated portion ofthe first example peptide array 400. Control peptide featuresalternately positioned with the first subset 404 include a second subset406, a third subset 408, and a fourth through a tenth subset 410-422,respectively. The first subset 404 was synthesized beginning during thefirst of twelve sequential synthesis periods, with the beginning ofsynthesis for each of the following subsets (i.e., the second subset 406through the tenth subset 422) being delayed by one synthesis periodrelative to the previously numbered subset. For example, the secondsubset 406 was synthesized beginning with the second synthesis period,the third subset 408 was synthesized beginning with the third synthesisperiod, and so forth, with the tenth subset 422 being synthesizedbeginning with the tenth synthesis period.

Following the completion of each of the twelve synthesis periods, eachof the control peptide features 402 were interrogated in the presence ofa receptor having an affinity for a binder sequence included in thecontrol peptide features. The receptor molecules were labelled anddetected to obtain a signal output indicative of the interaction betweenthe control peptide features and the receptor molecules. The resultingsignal was visualized both optically (FIG. 8) and graphically (FIG. 9)to determine the fidelity of synthesis of the control peptide features402, and by extension, the broader population of peptide features (notshown) on the peptide array 400. With respect to the optical imagingapproach, each of the control peptide features 402 are visually found tohave substantially the same signal output as represented by the degreeof shading of the boxes representing the control peptide features 402.It is noted that, with respect to FIGS. 8 and 10, a relatively darkershaded feature corresponds to a greater measured signal output, andtherefore, a greater degree of interaction between the particularpeptide feature and the receptor molecules. The measured signal outputfor each control peptide feature can also be plotted graphically, whichgenerally corresponds to the signal output as a function of location onthe surface of the peptide array 400. The graphical plot, as shown inFIG. 9 illustrates a trend similar to that observed in FIG. 8, whereeach of the control peptide features exhibits a uniform signal output.The signal output observed for the control peptide features 402 on thefirst example peptide array 400 is indicative of uniform synthesisbetween synthesis periods. Accordingly, it can be said that there islittle to no ‘drift’ in the quality or fidelity of synthesis over thecourse of the twelve synthesis periods synthesis operation.

Turning to FIGS. 10 and 11, a portion of a second example peptide array500 includes a plurality of control peptide features 502 synthesizedover twelve synthesis periods in a continuous uninterrupted pattern asdescribed for the peptide array 400. By way of comparison, a firstsubset 504 of the control peptide features 502 corresponds with thefirst subset 404, a second subset 506 corresponds with the second subset406, and so forth, with the third through tenth subsets 508-522corresponding to the third through tenth subsets 408-422. Following thecompletion of each of the twelve synthesis periods, each of the controlpeptide features 502 were interrogated as described above for thecontrol peptide features 402 to obtain a signal output indicative of theinteraction between the control peptide features 502 and the receptormolecules. The resulting signal was visualized both optically (FIG. 10)and graphically (FIG. 11) to determine the fidelity of synthesis of thecontrol peptide features 502, and by extension, the broader populationof peptide features (not shown) on the peptide array 500.

With respect to FIG. 10, each of the control peptide features 502 areobserved to have an increasing signal output as a function of thesynthesis period in which synthesis was initiated. For example, each ofthe illustrated features in the first subset 504 have the least shading(i.e., least signal output intensity), whereas the illustrated featureof tenth subset 522 has the darkest shading (i.e., greatest signaloutput intensity). Further, the shading increases with increasing subsetnumber from the first subset 504 to the tenth subset 522. With referenceto the graphical plot of measured signal output in FIG. 11, a trendsimilar to that observed in FIG. 10 further illustrates that each of thecontrol peptide features 502 exhibits a variable signal output with thesignal output increasing as a function of the synthesis period, withlater synthesized features having relatively greater signal outputintensity. The variable signal output observed for the control peptidefeatures 502 on the second example peptide array 500 is indicative ofnon-uniform synthesis between synthesis periods. Accordingly, it can besaid that there is drift in the quality or fidelity of synthesis overthe course of the twelve synthesis periods that make up the synthesisoperation.

In order to interpret the cause of the drift in the quality or fidelityof synthesis of a population of control peptide features, observationand analysis can be carried out for trends in the detected signal outputcharacteristic of interaction of the receptor molecules with theplurality of control peptide features. For example, an observation ofincreasing signal output associated with control peptide featuressynthesized beginning with later synthesis periods in a synthesisoperation (e.g., FIGS. 10 and 11) can indicate one or more possibleerrors or other adverse effects. In one aspect, the observed trend maybe indicative of a blockage in one of the reagent supply lines that wasslowly cleared over the course of the synthesis operation. The reducedsupply of reagent (e.g., one or more amino acids) to the peptide arraycan result in incompletely or inaccurately synthesized control peptidefeatures. In another aspect, the observed trend may indicate thatsynthesis reagents added in later synthesis periods were interactingwith already synthesized control peptide features. In this case, aminoacid reagents can react with side chains or other moieties associatedwith previously synthesized control peptide features, thereby reducingthe ability of the those features to bind or otherwise interact withreceptor molecules during downstream interrogation and detection steps.

In another example, an observation of decreasing signal outputassociated with control peptide features synthesized beginning withlater synthesis periods in a synthesis operation can indicate yet othererrors or other adverse effects. In one aspect, the observed trend maybe indicative of a blockage in one of the reagent supply lines thatdeveloped over the course of the synthesis operation. As discussedabove, the reduced supply of reagent (e.g., one or more amino acids) tothe peptide array can result in incomplete or inaccurate synthesis forcontrol peptide features synthesized during later synthesis periods.Still other errors and adverse effects are also possible. Accordingly,by first analyzing trends in signal output where there is drift presentin the corresponding peptide synthesis operation, and then identifyingthe cause of the trend, a database of correlations can be prepared. Overtime, the correlations can be recalled to quickly determine the cause(error or other adverse effect) associated with a particular trend insignal output data for a set of control peptide features on a peptidearray. Moreover, by determining the cause of drift or otherinconsistency in the signal output associated with control peptidefeatures, an assessment can be made of the quality or fidelity of thebroader population of peptide features (including the control peptidefeatures) on the same peptide array.

Whereas the hypothetical data presented in FIGS. 8-11 are for twoexample peptide arrays, actual signal output data followinginterrogation of a population of control peptide features can bepresented in alternative or additional formats. With reference to FIG.12, data was collected for a peptide array synthesis operation includingtwelve total synthesis periods. Control peptide features having thestreptavidin binder sequence WTHPQFE (SEQ ID NO:1) were synthesized overthree consecutive and uninterrupted synthesis periods (e.g., see FIG. 3)with control peptide features synthesized beginning with each of thefirst ten synthesis periods. Data included in the illustrated box plotchart corresponds to individual control peptide features within apopulation of control peptide features. The synthesis period in whichsynthesis began for each of the control peptide features is known (i.e.,predetermined based on the design for synthesis of the peptide array).The signal output is measured for each of the control peptide featuresusing one or more interrogation and detection techniques as describedherein. Signal output is then plotted as a function of the synthesisperiod in which a given control peptide feature was initiated withstatistical analysis performed on control peptide features synthesizedin parallel (i.e., during the same synthesis periods and synthesiscycles).

As shown in FIG. 12, a standard box and whisker plot can be used tovalidate quality control data associated with control peptide featuresfrom a given synthesis operation against aggregate quality control datacollected from a plurality of previous synthesis operations. In oneexample, data representing signal output as a function of receptorbinding to control peptides synthesized beginning in a particularsynthesis period can be combined from one or more synthesis operationsto graphically depict groups of numerical data through their quartiles(defined by the upper and lower bounds of the boxes). Lines extendingvertically from the boxes (whiskers) indicate variability outside theupper and lower quartiles. Individual data points for a given synthesisrun can then be plotted alongside the combined (aggregate) datarepresented by the boxes and whiskers.

Data in FIG. 12 for a set of control peptides synthesized in a firstsynthesis operation (open circles) is generally aligned (i.e., overlaps)with the boxes, which generally represent a signal output range that isindicative of successful control peptide synthesis. Accordingly, thefirst synthesis operation unconditionally passes the quality controlassessment. By contrast, data for a set of control peptides synthesizedin a second synthesis operation (open triangles) is generally alignedwith the boxes for each of the synthesis periods except for synthesisperiod four. As the low signal output measured for control peptidessynthesized beginning in synthesis period four of the second synthesisoperation is outside of the range indicated by both the box and whiskersin the corresponding period, the second synthesis operation fails thequality control assessment.

With continued reference to FIG. 12, each of the data points collectedfor both a third synthesis operation (filled squares) and a fourthsynthesis operation (filled diamonds) falls outside of the rangeindicated by the boxes and whiskers. With respect to the data for thethird synthesis operation, and taking into account the log-scale use forthe vertical axis, although the total signal is somewhat low relative tothe boxes and whiskers, the signal output is generally uniform at about2^(13.4) units over the course of the synthesis operation. The data forthe boxes and whiskers has a uniform signal output of about 2^(14.3)units over the course of the synthesis operation. In one aspect, thecontrol peptides in the third synthesis operation may conditionally passthe quality control assessment as a signal output of 2^(13.4) units isstill about 54% of 2^(14.3) units. However, it will be appreciated thatother factors or analyses can be relied upon when determining whetherthe third synthesis operation will pass or fail the quality controlassessment. With respect to the fourth set of control peptides, thesignal output in the initial synthesis period (denoted synthesis periodzero in FIG. 12) is about 2^(11.2) units. Thereafter the signal outputsteadily declines through synthesis period four, where the signal outputdrops to about 2^(9.9) units, which is about 5% of the signal output ofthe aggregate data (i.e., 2^(14.3) units). Given the decline in signalover the course of the several synthesis periods and the relatively lowsignal as a percentage of the aggregate data, the fourth synthesisoperation fails the quality control assessment.

VI. Method of Assessing Longitudinal Fidelity

Turning now to FIG. 13, a method 600 of assessing the fidelity of asynthetic peptide population includes a step 602 of creating an arraydesign. The array design can include specifications for a particularnumber of peptide features including one or more natural amino acids,non-natural amino acids, or other like monomer units. The array designcan further include specifications for the length of the one or morepeptide features, the total number of monomer units required, and soforth. Based on the specifications of the array design, a step 604 ofselecting representative peptide binders includes selection of at leastone peptide binder sequence. In one aspect, the peptide binder sequencecan be selected to include amino acids or other monomer units that arespecified for use in the array design. However, a binder sequence canadditionally or alternatively include monomer units that are not usedfor preparation of the peptide features specified in the step 602. Asdescribed above, one suitable peptide binder sequence includes thestreptavidin binder sequence WTHPQFE (SEQ ID NO:1). However, otherbinder sequences can suitably be selected. One possible criterion forselecting a suitable binder sequence includes the existence of adetectable reported molecule that is capable of interacting with thebinder sequence. Accordingly, it may be useful to select a bindersequence that is compatible with a known detection scheme.

In a next step 606, the number of synthesis cycles and synthesis periodsrequired to synthesize each of the peptide features in the array designis determined. In one aspect, the number of synthesis cycles can dependon the number of unique monomers required for synthesis of the peptidefeatures specified in the array design. For example, an array designincluding the twenty canonical amino acids may have at least twentysynthesis cycles with one synthesis cycle allocated for each of thetwenty canonical amino acids. In another aspect, the number of synthesisperiods required can depend on the number and order of synthesis cycles,the length of each of the peptide features, the complexity of thepeptide features (e.g., the number of unique monomers per feature), thelike, and combinations thereof.

Upon determining the number of synthesis cycles and synthesis periods inthe step 606, the minimum number of synthesis periods can be determinedfor synthesizing the binder sequence selected in the step 604. Forexample, the streptavidin binder sequence WTHPQFE (SEQ ID NO:1) can besynthesized in a minimum of three synthesis periods using the synthesisscheme illustrated in FIG. 3. Given the number of synthesis cycle andsynthesis periods, in a step 608 of the method 600, the control peptidesynthesis scheme is selected. The synthesis scheme relates to how theone or more control peptide features will be synthesized over the courseof the synthesis operation. For example, the control peptide featurescan be synthesized in a continuous uninterrupted manner with synthesisof a new control peptide feature initiated beginning in each of thefirst x-y synthesis periods, where x is the total number of synthesisperiods in the synthesis operation and y is the minimum number ofsynthesis periods required to completely synthesize the selected controlpeptide feature. Notably, one example of the aforementioned continuousuninterrupted synthesis scheme is described herein with respect to atleast FIG. 3. Other synthesis schemes can also be devised as describedherein. In one aspect, design of a synthesis scheme can depend onfactors such as the sequence of the control peptide features (e.g.,number of monomer units, composition of monomer units), the number ofsynthesis periods, the number of synthesis cycles per synthesis period,and the like.

In a step 610 of the method 600, a synthetic peptide population issynthesized using any suitable method, including those methods describedherein. The design of the synthetic peptide population includes theplurality of control peptide features where each of the control peptidesincludes one of the peptide binder sequences selected in the step 604.In one aspect, each of the control peptide features is synthesized tohave an amino acid sequence including a selected one of the bindersequences. However, it is anticipated that one or more synthesis errorsmay occur that will result in control peptides having a sequence thatdiffers from the selected peptide binder sequence. Errors that may occurduring synthesis can include mechanical failures that impact delivery ofthe various reagents to the peptide array, degradation of one or more ofthe reagents, and the like. For example, each of the amino acids usedfor peptide synthesis is delivered from a separate reservoir. If one ofthe fluid connections to an amino acid reservoir fails, or if the aminoacid reagent in the reservoir is degraded, then synthesis errors will bepresent for each peptide synthesized with the amino acid reagent inquestion. In certain situations, even though the error occurred, thepeptide array can still be generated with the errors remaining initiallyundetected. As a result, the actual control peptide sequence can differfrom the selected control peptide sequence.

In a next step 612 of the method 600, the synthetic peptide populationis interrogated in the presence of a receptor having an affinity for thepeptide binder sequences encoded by the control peptide features. In oneaspect, the step 612 can include contacting the population of peptideswith a plurality of receptor molecules (e.g., antibodies, peptides,proteins, enzymes, or the like). The receptor molecules can be unlabeledor labeled with a detectable tag such as a fluorescent marker. Inanother aspect, the step 612 can include labeling the receptor moleculeswith a detectable reporter molecule, such as a primary (and optionally asecondary) antibody, a dye, the like, or a combination thereof.Thereafter, in a step 614 of the method 600, an output of thepeptide-receptor interaction is detected. The step 614 can includedetecting the presence of the receptor using an optical technique (e.g.,absorbance, luminescence, reflectance, etc.), a chemical technique(e.g., enzymatic assays), or another suitable method of detecting asignal output characteristic of an interaction of the receptor with thecontrol peptides or control peptide features. In one aspect, the signaloutput is indicative of the fidelity of incorporation of a particularamino acid into a corresponding control peptide. Further, as theposition of the particular amino acid in the control peptide sequence isknown (i.e., the amino acid is at a defined position), it is furtherpossible to assess whether the position of the amino acid is correct.Accordingly, based on the output detected in the step 614, a step 616 ofthe method 600 can include assessing the longitudinal fidelity of aminoacid (or other like monomer) incorporation. That is, for a controlpeptide feature synthesized at a known location and over a knownselection of synthesis periods and synthesis cycles, the detectedinteraction of a receptor in the presence of the control peptide featureis indicative of whether there is variation or drift in synthesisfidelity within or between synthesis cycles, within or between synthesisperiods, or a combination thereof.

VII. Examples

In some embodiments, the present disclosure provides isolated artificialcontrol peptides with specific affinity to streptavidin. In thisembodiment, the disclosure includes peptides consisting of the sequenceWTHPQFE (SEQ ID NO:1). The disclosure further includes peptidescomprising alternative or additional binder sequences as described, forexample, in U.S. patent application Ser. No. 15/233,543 to Bannen etal., filed on 10 Aug. 2016. Moreover, shorter or longer peptides (e.g.,5, 6, 7, 8, 9, and up to 20 amino acids) comprising sequences disclosedherein and elsewhere are also part of the invention.

As discussed herein, a control peptide including (or consisting of)WTHPQFE (SEQ ID NO:1) can be used to identify the longitudinal qualityof a synthetic population of peptides including the control peptide. Inone aspect any substitution, insertion, modification, or deletion at anyposition within the control peptide can result in a reduction or loss ofsignal corresponding to an interaction between the control peptide andthe streptavidin receptor. Moreover, the reduction or loss of signal isdistinguishable relative to the signal produced from the interactionbetween the streptavidin receptor and the control peptide in terms ofboth raw signal and signal relative to a control peptide having thesubstitution, insertion, modification, or deletion.

The peptide binders specific for streptavidin can be used as qualitycontrol peptides for any application that is compatible with thedetection or capture of streptavidin, a fragment of streptavidin, or astreptavidin-biotin. However, other peptide binders can be similarlydeveloped for a given receptor molecule other than streptavidin.Moreover, two or more different peptide binders (i.e., binders thatdiffer in their amino acid sequence by at least one amino acid) that areeach specific for streptavidin can be used simultaneously in a givenpeptide array design. Alternatively (or in addition), two or moredifferent receptors can be used to detect one or more control peptidesequences.

In one example, a population of 2.88 million peptide features issynthesized on a 2.54 cm×7.62 cm array surface. Of the 2.88 millionfeatures synthesized, 228 of the features comprise control peptidesfeatures having the streptavidin peptide binder sequence for analysis oflongitudinal synthesis fidelity. The control peptide features aregrouped into blocks that are replicated at various locations across thearray surface. In the present example case, each block comprises 19control peptide features having the sequence WTHPQFE (SEQ ID NO:1). Thecontrol peptide features are arranges as shown in FIG. 1, such thatcontrol peptide features synthesized in an initial synthesis periods arealternated with control peptide features synthesized beginning in latersynthesis periods. Each block of control peptide features is repeated 12times across a single array for a total of 19×12 or 228 total controlpeptide features per array of 2.88 million peptide features. It will beappreciated, however, that a block of control peptides is not limited tothe sequences described herein, and more than one control peptidesequence may be included in a given design for a peptide array.

Control peptide features are analyzed following the completion of asynthesis operation by array deprotection, streptavidin binding, andarray scanning. Array deprotection to remove side-chain protectinggroups is performed in 95% trifluoroacetic acid (TFA) and 0.5%Triispropylsilane (TIPS) for 30 min. Arrays are then incubated twicesuccessively in methanol for 30 seconds each, rinsed four timessuccessively with reagent-grade water, and washed for one minute in TBST[1× Tris-buffered saline (TBS) and 0.05% Tween-20], and then washedtwice successively in TBS for a duration of one minute for each wash.Streptavidin binding is performed by incubating arrays in a streptavidinbath [0.005 mg/ml Cy5-labeled streptavidin, 1% alkali-soluble casein,0.5×TBS, and 0.05% Tween-20] for 1 hour at room temperature. Followingstreptavidin binding, arrays are washed twice in 1×TBS for a duration ofone minutes per wash, with a final wash (30 seconds) in reagent-gradewater. Streptavidin fluorescence signal is detected by scanning thearray at 2 μm resolution and 15% gain with a 635 nm excitationwavelength using an MS200 microarray scanner.

Box and whisker plots for analysis of control peptide feature data areprepared in a two-part process. First, data is aggregated for aplurality of synthesis operations that were individually determined topass a quality control assessment. Data was aggregated by calculatingthe median signal for peptides representing each synthesis period foreach of the synthesis operations to determine a reference distributionof median signals for each synthesis period. Second, data for a givensynthesis operation is collected and plotted alongside the aggregatedata plotted as described above. For example, each of the data pointsfor the synthesis operations in FIG. 12 represents the median signaloutput value of either twelve (12) control peptide features (synthesisperiods one through nine) or one hundred and twenty (120) controlpeptide features (synthesis period zero). Notably, the initial synthesisperiod (i.e., synthesis period zero) includes 10-fold more features thanthe remaining synthesis periods due to the design of the blocks ofcontrol peptide features as described above and shown, for example, inFIG. 1.

In the above example, each of the control peptide features wassynthesized to have the sequence WTHPQFE (SEQ ID NO:1). However, two ormore different control peptide features including different bindersequences can be synthesized on a peptide array during the samesynthesis operation. For example, it has been demonstrated that apeptide including the binder sequence FDEWL (SEQ ID NO:2) can be boundby streptavidin (see at least U.S. Patent Application PublicationUS2015/0185216 filed on 19 Dec. 2014 to Albert et al.). Accordingly, apeptide array synthesized according to the present disclosure caninclude a first population of control peptide features synthesized tohave an amino acid sequence including the binder sequence WTHPQFE (SEQID NO:1), and a second population of control peptide featuressynthesized to have an amino acid sequence including the binder sequenceFDEWL (SEQ ID NO:2). In one aspect, the binder sequence FDEWL (SEQ IDNO:2) does not include the “HPQ” motif present in the binder sequenceWTHPQFE (SEQ ID NO:1), and can therefore provide further informationuseful for analysis of control peptide feature data collected andvisualized as described herein. For example, it may be determined thatcontrol peptides including the binder sequence FDEWL (SEQ ID NO:2) weresuccessfully and accurately synthesized during one or more cycles orperiods in which control peptides including the binder sequence WTHPQFE(SEQ ID NO:1) were incorrectly or improperly synthesized (i.e., asdetermined by interrogation of the peptides with streptavidin). In thiscase, it may be possible to determine that an error occurred withrespect to one or more of the amino acids H, P or Q in control peptidesincluding the binder sequence WTHPQFE (SEQ ID NO:1), as none of theamino acids H, P and Q are included in the binder sequence FDEWL (SEQ IDNO:2).

The schematic flow charts shown in the Figures are generally set forthas logical flow chart diagrams. As such, the depicted order and labeledsteps are indicative of one embodiment of the presented method. Othersteps and methods may be conceived that are equivalent in function,logic, or effect to one or more steps, or portions thereof, of theillustrated method. Additionally, the format and symbols employed in theFigures are provided to explain the logical steps of the method and areunderstood not to limit the scope of the method. Although various arrowtypes and line types may be employed, they are understood not to limitthe scope of the corresponding method. Indeed, some arrows or otherconnectors may be used to indicate only the logical flow of the method.For instance, an arrow may indicate a waiting or monitoring period ofunspecified duration between enumerated steps of the depicted method.Additionally, the order in which a particular method occurs may or maynot strictly adhere to the order of the corresponding steps shown.

The present invention is presented in several varying embodiments in thefollowing description with reference to the Figures, in which likenumbers represent the same or similar elements. Reference throughoutthis specification to “one embodiment,” “an embodiment,” or similarlanguage means that a particular feature, structure, or characteristicdescribed in connection with the embodiment is included in at least oneembodiment of the present invention. Thus, appearances of the phrases“in one embodiment,” “in an embodiment,” and similar language throughoutthis specification may, but do not necessarily, all refer to the sameembodiment.

The described features, structures, or characteristics of the inventionmay be combined in any suitable manner in one or more embodiments. Inthe following description, numerous specific details are recited toprovide a thorough understanding of embodiments of the system. Oneskilled in the relevant art will recognize, however, that the system andmethod may both be practiced without one or more of the specificdetails, or with other methods, components, materials, and so forth. Inother instances, well-known structures, materials, or operations are notshown or described in detail to avoid obscuring aspects of theinvention. Accordingly, the foregoing description is meant to beexemplary, and does not limit the scope of present inventive concepts.

Each reference identified in the present application is hereinincorporated by reference in its entirety.

1. A method of assessing a synthetic peptide population, the methodcomprising: interrogating a population of peptide features in thepresence of a receptor having an affinity for a binder sequence, thepopulation of peptide features synthesized over a plurality of synthesisperiods, the population of peptide features including a plurality ofcontrol peptide features synthesized to have an amino acid sequenceincluding the binder sequence, the plurality of control peptide featuresincluding: a first control peptide feature synthesized beginning with afirst one of the plurality of synthesis periods; and a second controlpeptide feature synthesized beginning after the first one of theplurality of synthesis periods such that synthesis of the second controlpeptide feature is delayed by at least one synthesis period; anddetecting a signal output characteristic of an interaction of thereceptor with the plurality of control peptide features, the signaloutput indicative of the fidelity of synthesis of the population ofpeptide features.
 2. The method of claim 1, wherein each of theplurality of synthesis periods comprises a plurality of synthesiscycles, wherein each of the plurality of synthesis cycles corresponds tothe addition of a selected amino acid.
 3. The method of claim 1, whereinthe binder sequence is a streptavidin binder sequence, and the receptoris streptavidin.
 4. The method of claim 1, wherein the plurality ofcontrol peptides is synthesizable over a minimum number of synthesisperiods, and wherein at least a portion of the plurality of controlpeptides is synthesized over a number of synthesis periods greater thanthe minimum number of synthesis periods.
 5. The method of claim 4,wherein the minimum number of synthesis periods is at least twosynthesis periods.
 6. The method of claim 1, further comprising:contacting the population of peptide features in the presence of thereceptor with a fluorescent probe capable of binding to the receptor,and wherein the single output is a fluorescence intensity obtainedthrough fluorophore excitation-emission, the fluorescence intensityreflecting at least one of an abundance of a portion of the receptorassociated with the plurality of control peptide features and a bindingaffinity of the receptor to the plurality of control peptide features.7. The method of claim 1, wherein the population of peptide features iscovalently bound to a solid surface in an array.
 8. The method of claim7, wherein the peptide features are bound to the solid surface at adensity of at least about 100,000 features per square centimeter.
 9. Themethod of claim 1, wherein the output signal of the receptor is knownfor each of the plurality of binder sequences.
 10. The method of claim1, wherein the control peptide features are synthesized to have at leasta first amino acid sequence including a first binder sequence and asecond amino acid sequence including a second binder sequence differentfrom the first binder sequence, the receptor having an affinity for eachof the first binder sequence and the second binder sequence.
 11. Amethod of assessing the fidelity of a synthetic peptide population, themethod comprising synthesizing a population of peptide features on asolid surface over a plurality of sequential synthesis periods, thepopulation of peptide features comprising a plurality of sample peptidefeatures and a plurality of control peptide features synthesized to havean amino acid sequence including a binder sequence, the control peptidefeatures including: a first control peptide feature synthesizedbeginning with a first one of the plurality of synthesis periods; and asecond control peptide feature synthesized beginning after the first oneof the plurality of synthesis periods such that synthesis of the secondcontrol peptide feature is delayed by at least one synthesis period;contacting the population of peptide features on the solid surface witha receptor having an affinity for the binder sequence; and detecting anoutput characteristic of an interaction of the receptor with each of thecontrol peptide features, wherein the output is indicative of thelongitudinal fidelity of synthesis of the population of peptidefeatures.
 12. The method of claim 11, wherein each of the plurality ofsynthesis periods comprises a plurality of synthesis cycles, whereineach of the plurality of synthesis cycles corresponds to the addition ofa selected amino acid.
 13. The method of claim 11, wherein the bindersequence is a streptavidin binder sequence, and the receptor isstreptavidin.
 14. The method of claim 11, wherein the plurality ofcontrol peptides is synthesizable over a minimum number of synthesisperiods, and wherein at least a portion of the plurality of controlpeptides is synthesized over a number of synthesis periods greater thanthe minimum number of synthesis periods.
 15. The method of claim 11,further comprising: contacting the population of peptide features in thepresence of the receptor with a fluorescent probe capable of binding tothe receptor, wherein the signal output is a fluorescence intensityobtained through fluorophore excitation-emission, the fluorescenceintensity reflecting at least one of an abundance of a portion of thereceptor associated with the plurality of control peptide features and abinding affinity of the receptor to the plurality of control peptidefeatures.
 16. The method of claim 11, wherein each of the sample peptidefeatures has a defined sequence.
 17. The method of claim 16, wherein thepeptide features are bound to the solid surface at a density of at leastabout 100,000 features per square centimeter.
 18. The method of claim11, wherein the output signal of the receptor is known for each of theplurality of binder sequences.
 19. The method of claim 11, wherein thecontrol peptide features are synthesized to have at least a first aminoacid sequence including a first binder sequence and a second amino acidsequence including a second binder sequence different from the firstbinder sequence, the receptor having an affinity for each of the firstbinder sequence and the second binder sequence.
 20. A synthetic peptidearray, comprising: an array substrate comprising a solid support havinga reactive surface; and a population of peptide features immobilized onthe reactive surface, the population of peptide features synthesizedover a plurality of sequential synthesis periods, the population ofpeptide features including a plurality of control peptide featuressynthesized to have an amino acid sequence including a binder sequence,the plurality of control peptide features comprising: a first controlpeptide feature synthesized beginning with a first one of the pluralityof synthesis periods; and a second control peptide feature synthesizedbeginning after the first one of the plurality of synthesis periods suchthat synthesis of the second control peptide feature is delayed by atleast one synthesis period; wherein detecting a signal outputcharacteristic of an interaction of a receptor with each of the controlpeptide features is indicative of the fidelity of synthesis of thepopulation of peptide features.